Oxygen depletion deep within 3D tissue engineered scaffolds limits the viability of embedded cells, causing localized necrosis. The development of new biomaterials that can deliver oxygen locally to these cells in a physiological manner will mitigate oxygen depletion and necrosis in these cell populations. Our laboratories have recently been investigating in situ-forming protein hydrogels and novel mechanisms for globular protein self-assembly that utilize changes in electrostatic charges and hydrophobic interactions to form a hydrogel at physiological temperatures. Under the proper controlled pH conditions, the partially denatured proteins will maintain the necessary secondary structure that will be needed for function. This proposal will utilize this new understanding of induced protein self-assembly to develop a new albumin/myoglobin-based scaffold that can deliver oxygen on demand by neighboring cells. Albumin has been extensively characterized, is readily available via isolation from blood or via recombinant DNA technology, biocompatible, and has been used in medical devices such as vascular grafts while myoglobin is well characterized and readily available oxygen carrying globular protein. In order to rationally design the hydrogel for this purpose a combination of theoretical and experimental approaches will be used to evaluate these new materials while providing fundamental framework for understanding and designing better protein hydrogel materials. Based on preliminary results, we suspect there are fundamental limitations to the types and/or size of proteins that can be used as pH-induced hydrogel forming building blocks. To delineate those boundaries, we will a) perform a series of systematic atomistic molecular dynamics simulations to determine the structure of the pH denatured albumin and myoglobin, b) build coarse-grained models from the atomistic simulations, and perform Brownian Dynamics simulations to study gel formation and their mechanical properties for albumin and myoglobin, c) experimentally study the formation of the protein gels, measure their mechanical properties and test the predictions from the simulations, use these results for fine tuning of the model, d) predict and fabricate the optimal conditions for formation of denatured albumin based gels with functional myoglobin proteins, e) characterize the stability of incorporated myoglobin, and f) assess oxygen binding and release kinetics from the hydrogel. This research is a true integration of theoretical and experimental approaches to solve a problem: theoretical atomistic and coarse-grained protein representations in combination with superior computational power currently allow simulation of systems of biologically relevant size and timescale while advanced imaging and spectroscopic techniques enable direct comparison between simulations and experiments. The development of reliable and predictive models will enable the simulations to predict and guide the design of improved biomaterials. The techniques developed in this proposal lay the foundation for the design of additional functional protein hydrogel scaffolds for applications in areas such as wound healing and dialysis.